Downloaded from http://thorax.bmj.com/ on March 24, 2015 - Published by group.bmj.com
279
Thorax 1992;47:279-283
Effects of inhalation of N-formyl-methionylleucyl-phenylalanine in the well elderly and in patients with chronic bronchitis M J Peters, A B X Breslin, N Berend
Abstract
Background Inhalation of the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) produces bronchoconstriction in normal subjects. FMLP thus has a putative role as a mediator of bronchoconstriction associated with bacterial bronchial infection. Methods The effects of FMLP inhalation were examined in ten subjects with a history of chronic bronchitis and ten age matched control subjects. Each subject inhaled FMLP doses from 0 025 to 0-8 pmol to determine the provocative dose of FMLP causing a 20% fall in FEV5(PD20FMLP). FEVy was recorded every five minutes after the final FMLP inhalation until it had returned to 95% of baseline FEV, or 60 minutes had elapsed. The time to return to 95% baseline FEV, was recorded or extrapolated from the recovery curve as an index of rapidity of recovery. Total and differential white cell counts were performed on each subject at baseline and five and 15 minutes after
Department of Thoracic Medicine, Concord Hospital M J Peters
A B X Breslin Department of Medicine, Sydney University, Sydney, Australia N Berend Reprint requests to: Dr M J Peters, Department of Thoracic Medicine, National Heart and Lung Institute, London SW3 6LY, UK Accepted 10 December 1991
the final FMLP inhalation. Results The geometric mean PD20 FMLP in the patients with chronic bronchitis was 0'06 pmol (95% confidence interval 0-015-0-26), which was significantly lower than that in the control subjects (0-21 pmol (0 02-1P9)). PD20 FMLP in the patients with chronic bronchitis but not age matched controls (p = 0 35) was lower than that found previously in young normal subjects (0 35 pmol (0-071-8). The return to 95% baseline FEV, occurred after 86(10) minutes in subjects with chronic bronchitis and in 81(23) minutes in their age matched controls, in both cases being much slower than that seen in young subjects (29(9) minutes). Conclusion Patients with chronic bronchitis may be especially susceptible to formyl peptides elaborated by bacteria during bacterial bronchial infection. Bacterial bronchial infection is the commonest cause of acute respiratory failure in subjects with chronic airflow limitation.' 2 Mucosal oedema, mucus hypersecretion and mucus plugs may all contribute -to the increase in airway resistance. Release of specific mediators
by bacteria may cause histamine release3 or enhanced a adrenergic responsiveness.4 Formyl peptides such as N-formyl-
ethionyl-leucyl-phenylalanine (FMLP) are products of protein synthesis67 in proliferating bacterial8 and eukaryotic mitochondria.' FMLP contracts human bronchial smooth muscle in vitrol' and inhaled FMLP is a potent bronchoconstrictor in normal subjects." When nebulised intermittently for two hours in rabbits, FMLP produces marked airway inflammation.'2 FMLP may therefore be an important mediator of both bronchoconstriction and airway inflammation associated with bacterial bronchial infection. Patients with chronic bronchitis, in contrast to subjects with a similar smoking history but without sputum production, have mucosal and periglandular inflammation in large airways with neutrophil infiltration.'3 Although FMLP appears to have a direct contractile effect on human bronchial smooth muscle in vitro,'0 both neutrophils and macrophages release mediators in response to FMLP and these may contribute to bronchoconstriction in vivo.'4 FMLP induced bronchoconstriction in vivo in rabbits is partially dependent on neutrophils.'5 Increased local protease activity associated with airway inflammation may alter FMLP degradation and bioactivity. Thus there may be differences in the response to FMLP between normal young subjects and older subjects with chronic bronchitis, who are most susceptible to the development of respiratory failure during bacterial bronchial infection. The purpose of this study was to determine the effects of inhaled FMLP on subjects with chronic bronchitis and to compare changes in airway function and haematological changes after FMLP with age matched controls. Methods Ten subjects with a history of cough and sputum production consistent with the diagnosis of chronic bronchitis and ten age matched controls without chronic bronchitis were selected for the study. Subjects were recruited from patients referred for resection of lung cancer, except for three control subjects (one asymptomatic, one with atypical chest pain and one waiting for hip surgery). The control group consisted of six former smokers without chronic bronchitis and four subjects who had never smoked. Subjects in both groups were
Downloaded from http://thorax.bmj.com/ on March 24, 2015 - Published by group.bmj.com
280
Peters, Breslin, Berend
aged between 60 and 70 years and none had a history of asthma or recent respiratory infection. The presence or absence of chronic bronchitis and smoking history were determined by clinical interview before lung function testing and bronchial provocation. No subject was taking inhaled or oral corticosteroid or theophylline. Each subject performed a histamine challenge using the method of Yan.'6 The provocative dose of histamine needed to cause a 20% fall in FEV, (PD20 histamine) was always greater than 2 ,mol. The histamine challenge was performed on a separate day before the FMLP challenge. Subjects in the chronic bronchitis group, still smoking at the time of entry, were not challenged with FMLP or histamine until they had ceased smoking for seven days. The protocol was approved by the institutional ethics committee of Concord Hospital. All subjects gave informed consent for participation. FMLP PROVOCATION
FMLP (Sigma Chemicals) at a concentration of 5 mmol/l was dissolved in 50% dimethyl sulphoxide (DMSO)/saline and diluted with saline to produce doubling dilutions down to 150 imol/l. Five breaths of each concentration of solution were taken from a de Vilbiss 646 nebuliser powered by air at a flow rate of 8 1/min and pressure of 138 kPa, a French-Rosenthal dosimeter being used with duration of nebulisation 0-6 seconds. FEVy performed after inhalation of 25% DMSO/saline was used as control. The range of FMLP doses inhaled was 0-025-0-8 Mmol. FEVy was recorded three minutes after each inhalation, the endpoint of challenge being a 20% fall in FEV,. PD20 FMLP was determined by linear interpolation of the log-dose response curve. For statistical purposes subjects with a 20% fall in FEVy after the initial dose of FMLP were assigned a PD20 FMLP of 0-025 gmol; those who had a fall in FEVy of less than 20% after the largest dose of FMLP were assigned a PD20 FMLP of 0-8 imol. RECOVERY
FEVy was recorded at five minute intervals after the final dose of FMLP for 60 minutes or until FEV1 had returned to 95% of the baseline value. The rate of recovery from induced bronchoconstriction was assessed only in subjects who sustained a 20% or greater FEV1 fall in response to FMLP. The time to return to 95% of baseline FEV1 was determined by interpolation or linear extrapolation of the
FEV,-time curve. HISTAMINE RESPONSIVENESS
When assessment of recovery ceased (at 60 minutes or return to 95% baseline FEV,), a further histamine provocation test was performed. CIRCULATING LEUCOCYTES
Venous blood was taken at baseline and at five and 15 minutes after the final FMLP dose to determine the effect of FMLP inhalation on
circulating leucocyte count. The total white cell count was done on a Coulter counter and differential leucocyte count on 100 cells after staining with May-Grunwald-Giemsa. EFFECT OF THE DMSO DILUENT
Subjects with chronic bronchitis took six inhalations, each of five breaths of 25% DMSO/saline on a second day. FEVy was recorded two minutes after each inhalation. Histamine challenge was performed and blood taken at the same time as those on the FMLP day. The order in which DMSO and FMLP challenge was performed was randomised. DATA FROM YOUNG SUBJECTS
FMLP responsiveness and recovery from FMLP induced bronchoconstriction in young subjects was taken from results previously published.'" FMLP provocation and determination of recovery time was determined in identical fashion for these subjects except that the highest FMLP dose administered was 1-6 4umol rather than 0-8 /umol. ANALYSIS
Group comparisons were made between the subjects with chronic bronchitis, the age matched controls and previous data from normal young subjects." In examining values for PD20 FMLP from normal young subjects, those which were greater than 0-8 ,umol were censored to the maximum dose given in this study (0-8 imol). Log PD20 FMLP and time to return to baseline were compared by the Wilcoxon rank sum test. Comparison of PD20 histamine after FMLP and DMSO was made on log transformed data by the Wilcoxon test for paired differences. White cell and differential leucocyte counts were compared by using the Student's t test. Correlations were determined by nonparametric regression. Data are presented as mean (SE) or geometric mean (95 % confidence limits) as appropriate.
Results The mean age of the patients with chronic bronchitis was 66 years (range 62-70 years), compared with 65 years (range 63-69 years) in the elderly control subjects. The mean age of the young control subjects was 35 years (range 28-42 years). The mean (SE) baseline FEVy value in the patients with chronic bronchitis was 72(2 1)% of predicted, and in the elderly controls 82(5-3)% of predicted (p > 0-1). Mean (SE) baseline FEV1 in the young control subjects was 94(4-1)% of predicted, significantly greater than that in patients with chronic bronchitis but not elderly pontrols. Patients with chronic bronchitis had smoked a mean total of 59(3) pack years. The six former smokers in the age matched control group had smoked a mean of 55 pack years with a mean non-smoking interval of seven years (range 2-15 years). Baseline histamine responsiveness was similar in the two older groups. Geometric mean PD20 histamine in the patients with chronic bronchitis was 6-9 imol (4-7-10 2
Downloaded from http://thorax.bmj.com/ on March 24, 2015 - Published by group.bmj.com
281
Effects of inhalation of N-formyl-methionyl-leucyl-phenylalanine Figure I PD20FMLP illustratedfor subjects with chronic bronchitis (group 1), age matched control subjects (group 2) and young healthy subjects (group 3). Horizontal lines indicate geometric mean PD20FMLP in each group. Closed circles in groups 2 and 3 indicate lifelong non-smokers. PD20FMLP is significantly lower in the patients with chronic bronchitis than in both other groups. There is no difference between the older andyounger control
08
00000
.000
04
0
a
E
02
80
-I
0*1
0
0 0
0 -
0
O 005
.1.
0
0
a- 0.025-
0
0000
RECOVERY
Group 1
Group 2
Group3~
,umol) col pared withr6o 7 mol(range39-116
subjects.
young subjects and the older control group (95% confidence interval of the difference: -0-5-1-9 doubling dilutions). In the elderly control group there was no difference in PD20 FMLP between former smokers and those who had never smoked. There was no correlation in either group between log PD20 FMLP and baseline FEV1 or log PD20 histamine.
limol) in the control group.
All of the patients with chronic bronchitis and six of the older control subjects (three exsmokers and three who had never smoked) had a 20% or greater fall in FEV1 in response
to FMLP. In the patients with chronic bronchitis, mean (SEM) time to return to 95%
baseline FEVy was 86(10) minutes which was FMLP not significantly different from the 81(23) No subjoect in either older group exhibited minutes observed for the older control subjects facial flus;hing in response to FMLP. Geometric (figure 2). The duration of bronchoconstriction mean PD)20 FMLP in the patients with chronic was significantly longer in both older groups bronchitiis was 0-06 umol (0015-0-26 imol) than recovery in healthy younger subjects, in compare d with 0-21 jmol (0-02-1 9 pmol) in whom FEV, returned to 95% of baseline in the age miatched control subjects and 0-35 umol 29(9) minutes (table). (0-07-1 E3 ,mol) in the young normal subjects (figure 1 and table). The difference between EFFECT OF FMLP INHALATION ON HISTAMINE PD20 FMILP in patients with chronic bronchitis RESPONSIVENESS and you]ng normal subjects as 2-5 doubling Repeat histamine challenge in one subject with dilutions ,(95% confidence interval of the dif- chronic bronchitis was not possible after ference I1-7-3-2); that between patients with FMLP because FEV, was too low 60 minutes chronic E)ronchitis and their age matched con- after the final FMLP dose. Geometric mean trols wars 1-7 doubling dilutions (029-3 2). PD20 histamine in the nine subjects evaluable There wais no significant difference between the was 6-9 yimol (4-7-10-2 imol) at baseline, 4 6 jumol (1-3-18-0 pmol) after FMLP inhalation and 5-7 ,mol (3-0-10-9 ,umol) after DMSO. Characteristics of subjectsfor each group Neither FMLP nor DMSO produced a significant change in PD20 histamine. In the age matched control subjects PD20 histamine after Patient Age FEV, Sttoking PD,0 FMLP bDuration of FMLP inhalation was 4-0 umol (0-57-27-9 No Sex (years) (% pred) (p1 ack years) (pmol) (min) a value that also did not differ from limol), Group 1: Patients with chronic bronchitis baseline. 76 65 016 1 M 72 69 EFFECT 01F
2 3 4 5 6 7 8 9 10 Mean
M M M M M M F M F
64 62 69 69 70 64 64 64 66 66
76 63 73 75 57 62 83 76 84 72
Group 2: Elderly control subjects
1 2 3 4 5 6 7 8 9 10 Mean
M M F M M M M M F M
63 65 63 65 64 64 65 66 64 69 65
40 50 60 60 60 60 50 60 80
69 67 69 59 103 74 98 106 92 85 82
50 45 50 60 55 70 0 0 0 0
105 97 84 106 77
0 0 0 0 0 5 9 10 22 20
018 0o10
64 81
0025
156071
0-025 0-06
0025
0-025 006 > 0-8 > 0-8
0-04 0 03
>0-8
0-12 0-60 >0-8 0-10 0 05 0-21
143
72
63 86
M M F F M F
F M M M
38 40 29 30 40 29 30 31 42 40 35
111 82 100 76 97 94
1-2 0-4 0-28 0-8 1-6 0-12 > 1-6 0-16 0-04 > 1-6 0 35
The only change in circulating leucocyte count to achieve significance was a fall in monocyte count 15 minutes after the final FMLP dose in the patients with chronic bronchitis (figure 3). Two subjects in this group and five in the control group showed a fluctuation in either direction of greater than 25% of baseline
134 168
100
45 41
90
50 49 81
Group 3: Young subjects
I 2 3 4 5 6 7 8 9 10 Mean
EFFECT OF FMLP ON CIRCULATING LEUCOCYTES
9 90 15 24 21 18
21 33 29
.±
.0m 80 70 +
'L
60
50
3
10
20
30
40
50
60
Minutes after final FMLP dose 2 Figure Recovery from FMLP induced bronchoconstriction in all 10 bronchitic subjects (closed circles) and the six age matched control subjects (open circles) who had a 20% FEV, fall in response to FMLP.
Both groups recover more slowly than young subjects with similar initialfall in FEV,.
a
Downloaded from http://thorax.bmj.com/ on March 24, 2015 - Published by group.bmj.com
282
Peters, Breslin, Berend
explanation for the enhanced response to FMLP, a similarly increased response to histamine inhalation should have been present, 9 yet PD20 histamine was similar in the two older 6 groups of subjects. This suggests that some 6 other feature of chronic bronchitis leads to 4 enhanced FMLP responsiveness. Subjects with chronic bronchitis have airway 32 inflammation with an excess of neutrophils in ._________________________ .the mucosa.'3 Tissue localised neutrophils'8'9 Baseline 5 15 15 Baseline 5 and macrophages,20 which mediate FMLP induced bronchoconstriction in the guinea Monocytes Eosinophils pig,21 may be primed in the presence of inflam04 1.0 mation to produce an enhanced response to stimulation by FMLP. Alternatively, mucosal 08 0-3 which increases permeability to inflammation, 06 formyl peptides,22 may permit greater access 02 for FMLP across bronchial epithelium to 04 receptors within the submucosa, vasculature 0-1 and bronchial smooth muscle. Airway inflam0.2 mation in chronic bronchitis could modify the bronchoconstrictor response to FMLP by Baseline 5 15 Baseline 5 15 either mechanism. Recovery from FMLP induced bronchoconFigure 3 Mean (SEM) peripheral blood cell cou nts ( x 1 O/l) in patients with chronic striction was much slower in both older groups bronchits (closed circles) and well elderly control stubjects (open circles) at baseline and of subjects than in the younger subjects studied five and 15 minutes after the final FMLP inhalati(on. earlier. The observed difference was not dose related, as the young subjects, by virtue of their neutrophil cournt. Change in neutrophil count higher PD20 FMLP values, had inhaled more did not correlat ze with total FMLP dose. FMLP than either older group. Nor was it a function of a greater initial response to FMLP, EFFECTS OF THE: DMSO DILUENT as only subjects with a 20% or greater fall in Mean (SEM) F: EV, after inhalation of DMSO/ FEV, were compared. It is not a non-specific saline alone in t the subjects with chronic bron- effect of age as older subjects recover from chitis was 97 6( 1-0)% of the baseline value, the histamine induced bronchoconstriction at a greatest fall beiing 5-4%. PD20 histamine after rate similar to that of younger subjects (Peters, DMSO remainced within one doubling dilution unpublished observations). of the baseline Nvalue in all subjects. There was One explanation for this prolonged duration no change in t total or differential white cell of effect would be that metabolism of FMLP count. slows with age. In view of its distribution in the lung,23 FMLP hydrolysis in bronchial mucosa is likely to be via the enzyme neutral endopeptidase as in neutrophils.24 Inhibition of neutral Discussion Bacterial bron chial infection is the most endopeptidase enhances the effect of FMLP on common precip: itant of acute respiratory failure cholinergic neurotransmission in rabbit airway in subjects with chronic airflow limitation'2 and in vitro25 though we found a pronounced a major cause oif morbidity and mortality. We reduction in the bronchoconstrictor response found that sub Ijects with chronic bronchitis to FMLP inhalation after inhibition of neutral bronchoconstri(cted to lower doses of inhaled endopeptidase invivo.26Neutral endopeptidase FMLP and re covery from FMLP induced activity does not decline with age and, if bronchoconstril ction was delayed in the older anything, serum activity is increased modestly subjects irrespe ctive of the presence of chronic in subjects with chronic airflow limitation.27 The prolonged response to FMLP in the older bronchitis. There was n lo difference in PD20 FMLP, subjects cannot be explained by changes in recovery from bronchoconstriction or in the neutral endopeptidase. The neutrophil dependent oedema seen in haematological effect of FMLP inhalation between the exc-smokers and those who had rabbit skin after FMLP injection is markedly never smoked iin the control group, so we feel accentuated by vasodilator pretreatment and that they repres gent a valid control group. The has a halflife of 40 minutes.28 Persistence of differences in IFMLP responses between the airway oedema could explain the prolonged bronchitic sut bjects and the ex-smoking effect of FMLP in older subjects but whether controls occurre .d despite a close match for total clearance of oedema slows with age or smoking historry. This implies that excess bronchoconstriction is related more closely to sputum producition predicts greater sensitivity airway oedema in this group is unknown. to FMLP inhal; ation. Leucocyte depletion'5 and anticholinergic Small airwalys of smokers with airflow agents23 inhibit FMLP induced bronchoconobstruction hav7e thickened walls and smaller striction but there is no evidence of differences luminal diame ters than those of control in the cholinergic or leucocyte response to subjects.'7 As a result, the reduction in airflow FMLP in this age group to account for the would be expectted to be greater in patients with slower recovery. FMLP inhalation did not produce changes chronic bronchiitis for an equivalent degree of smooth muscle contraction. If this was the sole in neutrophil or monocyte counts in venous White cells
12
Neutrophils
Downloaded from http://thorax.bmj.com/ on March 24, 2015 - Published by group.bmj.com
Effects of inhalation of N-formyl-methionyl-leucyl-phenylalanine
blood in either group of the magnitude that we had seen in normal subjects.29 There was also a total absence of facial flushing in both older groups after FMLP inhalation whereas flushing is seen in over 75% of young subjects,""293' always occurring after the first FMLP dose and always associated with marked neutropenia.29 Transient neutropenia after FMLP inhalation probably reflects synchronous activation of circulating neutrophils and temporary sequestration in the microcirculation. We have usually given a single inhalation of 0-4 jumol FMLP to young subjects after finding variability in the neutrophil responses to incremental doses of FMLP (0 1, 0-5, 1 Mmol).3` Nevertheless, all young normal subjects inhaling the incremental doses of FMLP had either a transient neutropenia or neutrophilia or both; in this study even the five older control subjects who had a dose of FMLP equivalent to that given to young normal subjects had a minimal neutrophil leucocytosis following FMLP. The leucocyte response to FMLP may require access of FMLP to the circulation. Neutrophils from well older subjects have higher resting intracellular calcium concentrations and the response to a variety of stimuli including FMLP is slightly reduced.32 33 A final possibility is that the neutrophils of older subjects, behave in a similar way to those of younger subjects but less in synchrony so that flushing and profound neutropenia which may require synchronous neutrophil activation adherence are not seen. Although the neutropenic response was absent in the older subjects, bronchoconstriction was similar in the older control subjects to that in the younger normal subjects and was increased in subjects with chronic bronchitis. The role that leucocytes play in FMLP induced bronchoconstriction is clearly not linked directly to the development of neutropenia in either age group. In this study, subjects with chronic bronchitis showed greater sensitivity to the effects of inhaled FMLP and older subjects showed delayed recovery from FMLP induced bronchoconstriction in comparison with young subjects. The combination of disease related increased sensitivity and age related slowing of recovery would place an older patient with chronic bronchitis at special risk ofthe effects of FMLP and related formyl peptides produced by bacteria during bacterial bronchial infection. We thank Boehringer Ingelheim and the Asthma Foundation of New South Wales for their support. 1 Burk RH, George RB. Acute respiratory failure in chronic obstructive pulmonary disease. Arch Intern Med 1973; 132:865-8. 2 Dardes N, Campo S, Chiappini MG, Re MA, Cicciarell P, Vulterini S. Prognosis of COPD patients after an episode of acute respiratory failure. Eur J Respir Dis 1986;69 (Suppl 146).377-81. 3 Martin RR, White A. The in vitro release of leukocyte histamine by staphylococcal antigens. J Immunol 1969; 102:437-41. 4 Van der Zwan JC, Orie NGM, Kauffman HF, Wiers PWJ, De Vries K. Bronchial obstructive reactions after inhalation with endotoxin and precipitogens of Haemophilus influenzae in patients with chronic non-specific lung disease. Clin Allergy 1982;12:547-9. 5 Simonsson BG, Svedmyr N, Skoogh BE, Andersson R, Bergh NP. In vivo and in vitro studies on alpha-receptors in human airways; potentiation with bacterial endotoxin. Scand J Respir Dis 1972;53:227-36.
283 6 Adams JM, Capecchi MR. N-formylmethionyl-sRNA as the initiator of protein synthesis. Proc Natl Acad Sci USA 1966;55: 147-54. 7 Capecchi MR. Initiation of E. coli proteins. Proc Natl Acad Sci USA 1966;55:1517-24. 8 Becker EL. The formylpeptide receptor of the neutrophil. Am JPathol 1987;129:16-24. 9 Carp H. Mitochondrial N-formylmethionyl proteins as chemoattractants for neutrophils. J Exp Med 1982;155: 264-75. 10 Armour CL, Black JL, Johnson PRA, Vincenc KS, Berend N. Formylpeptide induced contraction of human airways in vitro. J Appl Physiol 1986;60:141-6. 11 Berend N, Peters MJ, Armour CL, Black JL, Ward HE. Effect of formyl-methionyl-leucyl-phenylalanine on airway function. Thorax 1988;42:36-40. 12 Peters MJ, Panaretto K, Breslin ABX, Berend N. Effects of prolonged inhalation of N-formyl-methionyl-leucylphenylalanine on airway function in rabbits. J Appl Physiol 1991;70:2448-54. 13 Mullen JBM, Wright JL, Wiggs BR, Pare PD, Hogg JC. Reassessment of inflammation in airways in chronic bronchitis. BMJ 1985;291:1235-40. 14 Jubiz W, Radmark C, Malmsten C, et al. A novel leukotriene produced by stimulation of leukocytes with formylmethionylleucylphenylalanine. J Biol Chem 1982;257: 6106-10. 15 Berend N, Armour CL, Black JL. Formyl-methionylleucyl-phenylalanine causes bronchocontriction in rabbits. Agents Actions 1986;17:466-71. 16 Yan K, Salome C, Woolcock AJ. Rapid method for measurement of histamine responsiveness. Thorax 1983; 38:760-5. 17 Bosken C, Wiggs B, Pare P, Hogg J. Small airways dimensions in smokers with obstruction to airflow. Am Rev Respir Dis 1990;142:563-70. 18 Zimmerli W, Seligmann BE, Gallin JI. Neutrophils are hyperpolarised after exudation and show an increased response to formyl-peptide but not to phorbol myristyl acetate. Eur J Clin Invest 1987;17:435-41. 19 Zimmerli W, Seligmann BE, Gallin JI. Exudation primes human and guinea-pig neutrophils for subsequent responsiveness to the chemotactic peptide fMet-Leu-Phe and increases C3bi receptor expression. J Clin Invest 1986;77:925-33. 20 Williams JD, Robin J-L, Lewis RA, Lee TH, Austen KF. Generation of leukotrienes by human monocytes pretreated with cytochalasin B and stimulated with formylmethionyl-leucyl-phenyalalanine. J Immunol 1986;136: 642-8. 21 Boukili MA, Bureau M-F, Lellouch-Tubiana A, Lefort J, Simon M-T, Varhaftig BB. Alveolar macrophages and eicosanoids but not neutrophils mediate bronchoconstriction induced by FMLP in the guinea-pig. Br J Pharmacol 1989;98:61-70. 22 Hobson CH, Butt TJ, Ferry DM, Hunter J, Chadwick VS, Broom MF. Enterohepatic recirculation of bacterial chemotactic peptide in rats with experimental colitis. Gastroenterology 1988;94:1006-13. 23 Llorens C, Schwartz J-C. Enkephalinase activity in rat peripheral organs. Eur J Pharmacol 1981;69:113-16. 24 Connelly JC, Skidgel RA, Schulz WW, Johnson AR, Erdos EG. Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide. Proc Natl Acad Sci USA 1985;82:8737-41. 25 Kanemura T, Tamaoki J, Sakai N, Kobayashi K, Takizawa T. The effect ofN-formyl-methionyl-leucylphenylalanine on cholinergic neurotransmission and its modulation by enkephalinase in rabbit airway smooth muscle. Reg Pept 1989;26: 107-16. 26 Peters MJ, Panaretto K, Breslin ABX, Berend N. Effect of neutral endopeptidase inhibition on f-Met-Leu-Phe induced bronchoconstriction in the rabbit. J Appl Physiol 1991;70:877-81. 27 Johnson AR, Coalson JL, Ashton J, Larumbride M, Erdos EG. Neutral endopeptidase in serum samples from patients with adult respiratory distress syndrome. Comparison with angiotensin converting enzyme. Am Rev Respir Dis 1985;132:1262-7. 28 Hellewell PG, Yarwood H, Williams TJ. Characteristics of oedema formation induced by N-formyl-methionylleucyl-phenylalanine in rabbit skin. Br J Pharmacol 1989; 97:181-9. 29 Peters MJ, Breslin ABX, Kemp AS, Chu J, Berend N. The hematologicaleffectsofinhalationofN-formyl-methionylleucyl-phenylalanine in man. Thorax 1992;47:284-7. 30 Peters MJ, Breslin ABX, Berend N. The effect of anticholinergic and beta-agonist pretreatment on bronchoconstriction induced by N-formyl-methionyl-leucylphenylalanine. Fur Respir I 1989,2.464-9. 31 Peters MJ, Karet B, Breslin ABX, Berend N. Histamine reactivity and hematologic changes following N-formylmethionyl-leucyl-phenylalanine (FMLP) inhalation in man [abstract]. Aust NZ J Med 1987;17(Suppl):520. 32 Varga Z, Kovacs EM, Paragh G, Jacob M-P, Robert L, Fulop T. Effects of elastin peptides and N-formylmethionyl-leucyl-phenyalanine on cytosolic free calcium in polymorphonuclear leukocytes in healthy middle-aged and elderly subjects. Clin Biochem 1988;21:127-30. 33 Lipschitz DA, Udupa KB, Boxer LA. The role ofcalcium in the age related decline of neutrophil function. Blood 1988;71:659-65.
Downloaded from http://thorax.bmj.com/ on March 24, 2015 - Published by group.bmj.com
Effects of inhalation of N-formyl-methionyl-leucyl-phenylalanine in the well elderly and in patients with chronic bronchitis. M J Peters, A B Breslin and N Berend Thorax 1992 47: 279-283
doi: 10.1136/thx.47.4.279 Updated information and services can be found at: http://thorax.bmj.com/content/47/4/279
These include:
Email alerting service
Receive free email alerts when new articles cite this article. Sign up in the box at the top right corner of the online article.
Notes
To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions To order reprints go to: http://journals.bmj.com/cgi/reprintform To subscribe to BMJ go to: http://group.bmj.com/subscribe/
279
Thorax 1992;47:279-283
Effects of inhalation of N-formyl-methionylleucyl-phenylalanine in the well elderly and in patients with chronic bronchitis M J Peters, A B X Breslin, N Berend
Abstract
Background Inhalation of the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) produces bronchoconstriction in normal subjects. FMLP thus has a putative role as a mediator of bronchoconstriction associated with bacterial bronchial infection. Methods The effects of FMLP inhalation were examined in ten subjects with a history of chronic bronchitis and ten age matched control subjects. Each subject inhaled FMLP doses from 0 025 to 0-8 pmol to determine the provocative dose of FMLP causing a 20% fall in FEV5(PD20FMLP). FEVy was recorded every five minutes after the final FMLP inhalation until it had returned to 95% of baseline FEV, or 60 minutes had elapsed. The time to return to 95% baseline FEV, was recorded or extrapolated from the recovery curve as an index of rapidity of recovery. Total and differential white cell counts were performed on each subject at baseline and five and 15 minutes after
Department of Thoracic Medicine, Concord Hospital M J Peters
A B X Breslin Department of Medicine, Sydney University, Sydney, Australia N Berend Reprint requests to: Dr M J Peters, Department of Thoracic Medicine, National Heart and Lung Institute, London SW3 6LY, UK Accepted 10 December 1991
the final FMLP inhalation. Results The geometric mean PD20 FMLP in the patients with chronic bronchitis was 0'06 pmol (95% confidence interval 0-015-0-26), which was significantly lower than that in the control subjects (0-21 pmol (0 02-1P9)). PD20 FMLP in the patients with chronic bronchitis but not age matched controls (p = 0 35) was lower than that found previously in young normal subjects (0 35 pmol (0-071-8). The return to 95% baseline FEV, occurred after 86(10) minutes in subjects with chronic bronchitis and in 81(23) minutes in their age matched controls, in both cases being much slower than that seen in young subjects (29(9) minutes). Conclusion Patients with chronic bronchitis may be especially susceptible to formyl peptides elaborated by bacteria during bacterial bronchial infection. Bacterial bronchial infection is the commonest cause of acute respiratory failure in subjects with chronic airflow limitation.' 2 Mucosal oedema, mucus hypersecretion and mucus plugs may all contribute -to the increase in airway resistance. Release of specific mediators
by bacteria may cause histamine release3 or enhanced a adrenergic responsiveness.4 Formyl peptides such as N-formyl-
ethionyl-leucyl-phenylalanine (FMLP) are products of protein synthesis67 in proliferating bacterial8 and eukaryotic mitochondria.' FMLP contracts human bronchial smooth muscle in vitrol' and inhaled FMLP is a potent bronchoconstrictor in normal subjects." When nebulised intermittently for two hours in rabbits, FMLP produces marked airway inflammation.'2 FMLP may therefore be an important mediator of both bronchoconstriction and airway inflammation associated with bacterial bronchial infection. Patients with chronic bronchitis, in contrast to subjects with a similar smoking history but without sputum production, have mucosal and periglandular inflammation in large airways with neutrophil infiltration.'3 Although FMLP appears to have a direct contractile effect on human bronchial smooth muscle in vitro,'0 both neutrophils and macrophages release mediators in response to FMLP and these may contribute to bronchoconstriction in vivo.'4 FMLP induced bronchoconstriction in vivo in rabbits is partially dependent on neutrophils.'5 Increased local protease activity associated with airway inflammation may alter FMLP degradation and bioactivity. Thus there may be differences in the response to FMLP between normal young subjects and older subjects with chronic bronchitis, who are most susceptible to the development of respiratory failure during bacterial bronchial infection. The purpose of this study was to determine the effects of inhaled FMLP on subjects with chronic bronchitis and to compare changes in airway function and haematological changes after FMLP with age matched controls. Methods Ten subjects with a history of cough and sputum production consistent with the diagnosis of chronic bronchitis and ten age matched controls without chronic bronchitis were selected for the study. Subjects were recruited from patients referred for resection of lung cancer, except for three control subjects (one asymptomatic, one with atypical chest pain and one waiting for hip surgery). The control group consisted of six former smokers without chronic bronchitis and four subjects who had never smoked. Subjects in both groups were
Downloaded from http://thorax.bmj.com/ on March 24, 2015 - Published by group.bmj.com
280
Peters, Breslin, Berend
aged between 60 and 70 years and none had a history of asthma or recent respiratory infection. The presence or absence of chronic bronchitis and smoking history were determined by clinical interview before lung function testing and bronchial provocation. No subject was taking inhaled or oral corticosteroid or theophylline. Each subject performed a histamine challenge using the method of Yan.'6 The provocative dose of histamine needed to cause a 20% fall in FEV, (PD20 histamine) was always greater than 2 ,mol. The histamine challenge was performed on a separate day before the FMLP challenge. Subjects in the chronic bronchitis group, still smoking at the time of entry, were not challenged with FMLP or histamine until they had ceased smoking for seven days. The protocol was approved by the institutional ethics committee of Concord Hospital. All subjects gave informed consent for participation. FMLP PROVOCATION
FMLP (Sigma Chemicals) at a concentration of 5 mmol/l was dissolved in 50% dimethyl sulphoxide (DMSO)/saline and diluted with saline to produce doubling dilutions down to 150 imol/l. Five breaths of each concentration of solution were taken from a de Vilbiss 646 nebuliser powered by air at a flow rate of 8 1/min and pressure of 138 kPa, a French-Rosenthal dosimeter being used with duration of nebulisation 0-6 seconds. FEVy performed after inhalation of 25% DMSO/saline was used as control. The range of FMLP doses inhaled was 0-025-0-8 Mmol. FEVy was recorded three minutes after each inhalation, the endpoint of challenge being a 20% fall in FEV,. PD20 FMLP was determined by linear interpolation of the log-dose response curve. For statistical purposes subjects with a 20% fall in FEVy after the initial dose of FMLP were assigned a PD20 FMLP of 0-025 gmol; those who had a fall in FEVy of less than 20% after the largest dose of FMLP were assigned a PD20 FMLP of 0-8 imol. RECOVERY
FEVy was recorded at five minute intervals after the final dose of FMLP for 60 minutes or until FEV1 had returned to 95% of the baseline value. The rate of recovery from induced bronchoconstriction was assessed only in subjects who sustained a 20% or greater FEV1 fall in response to FMLP. The time to return to 95% of baseline FEV1 was determined by interpolation or linear extrapolation of the
FEV,-time curve. HISTAMINE RESPONSIVENESS
When assessment of recovery ceased (at 60 minutes or return to 95% baseline FEV,), a further histamine provocation test was performed. CIRCULATING LEUCOCYTES
Venous blood was taken at baseline and at five and 15 minutes after the final FMLP dose to determine the effect of FMLP inhalation on
circulating leucocyte count. The total white cell count was done on a Coulter counter and differential leucocyte count on 100 cells after staining with May-Grunwald-Giemsa. EFFECT OF THE DMSO DILUENT
Subjects with chronic bronchitis took six inhalations, each of five breaths of 25% DMSO/saline on a second day. FEVy was recorded two minutes after each inhalation. Histamine challenge was performed and blood taken at the same time as those on the FMLP day. The order in which DMSO and FMLP challenge was performed was randomised. DATA FROM YOUNG SUBJECTS
FMLP responsiveness and recovery from FMLP induced bronchoconstriction in young subjects was taken from results previously published.'" FMLP provocation and determination of recovery time was determined in identical fashion for these subjects except that the highest FMLP dose administered was 1-6 4umol rather than 0-8 /umol. ANALYSIS
Group comparisons were made between the subjects with chronic bronchitis, the age matched controls and previous data from normal young subjects." In examining values for PD20 FMLP from normal young subjects, those which were greater than 0-8 ,umol were censored to the maximum dose given in this study (0-8 imol). Log PD20 FMLP and time to return to baseline were compared by the Wilcoxon rank sum test. Comparison of PD20 histamine after FMLP and DMSO was made on log transformed data by the Wilcoxon test for paired differences. White cell and differential leucocyte counts were compared by using the Student's t test. Correlations were determined by nonparametric regression. Data are presented as mean (SE) or geometric mean (95 % confidence limits) as appropriate.
Results The mean age of the patients with chronic bronchitis was 66 years (range 62-70 years), compared with 65 years (range 63-69 years) in the elderly control subjects. The mean age of the young control subjects was 35 years (range 28-42 years). The mean (SE) baseline FEVy value in the patients with chronic bronchitis was 72(2 1)% of predicted, and in the elderly controls 82(5-3)% of predicted (p > 0-1). Mean (SE) baseline FEV1 in the young control subjects was 94(4-1)% of predicted, significantly greater than that in patients with chronic bronchitis but not elderly pontrols. Patients with chronic bronchitis had smoked a mean total of 59(3) pack years. The six former smokers in the age matched control group had smoked a mean of 55 pack years with a mean non-smoking interval of seven years (range 2-15 years). Baseline histamine responsiveness was similar in the two older groups. Geometric mean PD20 histamine in the patients with chronic bronchitis was 6-9 imol (4-7-10 2
Downloaded from http://thorax.bmj.com/ on March 24, 2015 - Published by group.bmj.com
281
Effects of inhalation of N-formyl-methionyl-leucyl-phenylalanine Figure I PD20FMLP illustratedfor subjects with chronic bronchitis (group 1), age matched control subjects (group 2) and young healthy subjects (group 3). Horizontal lines indicate geometric mean PD20FMLP in each group. Closed circles in groups 2 and 3 indicate lifelong non-smokers. PD20FMLP is significantly lower in the patients with chronic bronchitis than in both other groups. There is no difference between the older andyounger control
08
00000
.000
04
0
a
E
02
80
-I
0*1
0
0 0
0 -
0
O 005
.1.
0
0
a- 0.025-
0
0000
RECOVERY
Group 1
Group 2
Group3~
,umol) col pared withr6o 7 mol(range39-116
subjects.
young subjects and the older control group (95% confidence interval of the difference: -0-5-1-9 doubling dilutions). In the elderly control group there was no difference in PD20 FMLP between former smokers and those who had never smoked. There was no correlation in either group between log PD20 FMLP and baseline FEV1 or log PD20 histamine.
limol) in the control group.
All of the patients with chronic bronchitis and six of the older control subjects (three exsmokers and three who had never smoked) had a 20% or greater fall in FEV1 in response
to FMLP. In the patients with chronic bronchitis, mean (SEM) time to return to 95%
baseline FEVy was 86(10) minutes which was FMLP not significantly different from the 81(23) No subjoect in either older group exhibited minutes observed for the older control subjects facial flus;hing in response to FMLP. Geometric (figure 2). The duration of bronchoconstriction mean PD)20 FMLP in the patients with chronic was significantly longer in both older groups bronchitiis was 0-06 umol (0015-0-26 imol) than recovery in healthy younger subjects, in compare d with 0-21 jmol (0-02-1 9 pmol) in whom FEV, returned to 95% of baseline in the age miatched control subjects and 0-35 umol 29(9) minutes (table). (0-07-1 E3 ,mol) in the young normal subjects (figure 1 and table). The difference between EFFECT OF FMLP INHALATION ON HISTAMINE PD20 FMILP in patients with chronic bronchitis RESPONSIVENESS and you]ng normal subjects as 2-5 doubling Repeat histamine challenge in one subject with dilutions ,(95% confidence interval of the dif- chronic bronchitis was not possible after ference I1-7-3-2); that between patients with FMLP because FEV, was too low 60 minutes chronic E)ronchitis and their age matched con- after the final FMLP dose. Geometric mean trols wars 1-7 doubling dilutions (029-3 2). PD20 histamine in the nine subjects evaluable There wais no significant difference between the was 6-9 yimol (4-7-10-2 imol) at baseline, 4 6 jumol (1-3-18-0 pmol) after FMLP inhalation and 5-7 ,mol (3-0-10-9 ,umol) after DMSO. Characteristics of subjectsfor each group Neither FMLP nor DMSO produced a significant change in PD20 histamine. In the age matched control subjects PD20 histamine after Patient Age FEV, Sttoking PD,0 FMLP bDuration of FMLP inhalation was 4-0 umol (0-57-27-9 No Sex (years) (% pred) (p1 ack years) (pmol) (min) a value that also did not differ from limol), Group 1: Patients with chronic bronchitis baseline. 76 65 016 1 M 72 69 EFFECT 01F
2 3 4 5 6 7 8 9 10 Mean
M M M M M M F M F
64 62 69 69 70 64 64 64 66 66
76 63 73 75 57 62 83 76 84 72
Group 2: Elderly control subjects
1 2 3 4 5 6 7 8 9 10 Mean
M M F M M M M M F M
63 65 63 65 64 64 65 66 64 69 65
40 50 60 60 60 60 50 60 80
69 67 69 59 103 74 98 106 92 85 82
50 45 50 60 55 70 0 0 0 0
105 97 84 106 77
0 0 0 0 0 5 9 10 22 20
018 0o10
64 81
0025
156071
0-025 0-06
0025
0-025 006 > 0-8 > 0-8
0-04 0 03
>0-8
0-12 0-60 >0-8 0-10 0 05 0-21
143
72
63 86
M M F F M F
F M M M
38 40 29 30 40 29 30 31 42 40 35
111 82 100 76 97 94
1-2 0-4 0-28 0-8 1-6 0-12 > 1-6 0-16 0-04 > 1-6 0 35
The only change in circulating leucocyte count to achieve significance was a fall in monocyte count 15 minutes after the final FMLP dose in the patients with chronic bronchitis (figure 3). Two subjects in this group and five in the control group showed a fluctuation in either direction of greater than 25% of baseline
134 168
100
45 41
90
50 49 81
Group 3: Young subjects
I 2 3 4 5 6 7 8 9 10 Mean
EFFECT OF FMLP ON CIRCULATING LEUCOCYTES
9 90 15 24 21 18
21 33 29
.±
.0m 80 70 +
'L
60
50
3
10
20
30
40
50
60
Minutes after final FMLP dose 2 Figure Recovery from FMLP induced bronchoconstriction in all 10 bronchitic subjects (closed circles) and the six age matched control subjects (open circles) who had a 20% FEV, fall in response to FMLP.
Both groups recover more slowly than young subjects with similar initialfall in FEV,.
a
Downloaded from http://thorax.bmj.com/ on March 24, 2015 - Published by group.bmj.com
282
Peters, Breslin, Berend
explanation for the enhanced response to FMLP, a similarly increased response to histamine inhalation should have been present, 9 yet PD20 histamine was similar in the two older 6 groups of subjects. This suggests that some 6 other feature of chronic bronchitis leads to 4 enhanced FMLP responsiveness. Subjects with chronic bronchitis have airway 32 inflammation with an excess of neutrophils in ._________________________ .the mucosa.'3 Tissue localised neutrophils'8'9 Baseline 5 15 15 Baseline 5 and macrophages,20 which mediate FMLP induced bronchoconstriction in the guinea Monocytes Eosinophils pig,21 may be primed in the presence of inflam04 1.0 mation to produce an enhanced response to stimulation by FMLP. Alternatively, mucosal 08 0-3 which increases permeability to inflammation, 06 formyl peptides,22 may permit greater access 02 for FMLP across bronchial epithelium to 04 receptors within the submucosa, vasculature 0-1 and bronchial smooth muscle. Airway inflam0.2 mation in chronic bronchitis could modify the bronchoconstrictor response to FMLP by Baseline 5 15 Baseline 5 15 either mechanism. Recovery from FMLP induced bronchoconFigure 3 Mean (SEM) peripheral blood cell cou nts ( x 1 O/l) in patients with chronic striction was much slower in both older groups bronchits (closed circles) and well elderly control stubjects (open circles) at baseline and of subjects than in the younger subjects studied five and 15 minutes after the final FMLP inhalati(on. earlier. The observed difference was not dose related, as the young subjects, by virtue of their neutrophil cournt. Change in neutrophil count higher PD20 FMLP values, had inhaled more did not correlat ze with total FMLP dose. FMLP than either older group. Nor was it a function of a greater initial response to FMLP, EFFECTS OF THE: DMSO DILUENT as only subjects with a 20% or greater fall in Mean (SEM) F: EV, after inhalation of DMSO/ FEV, were compared. It is not a non-specific saline alone in t the subjects with chronic bron- effect of age as older subjects recover from chitis was 97 6( 1-0)% of the baseline value, the histamine induced bronchoconstriction at a greatest fall beiing 5-4%. PD20 histamine after rate similar to that of younger subjects (Peters, DMSO remainced within one doubling dilution unpublished observations). of the baseline Nvalue in all subjects. There was One explanation for this prolonged duration no change in t total or differential white cell of effect would be that metabolism of FMLP count. slows with age. In view of its distribution in the lung,23 FMLP hydrolysis in bronchial mucosa is likely to be via the enzyme neutral endopeptidase as in neutrophils.24 Inhibition of neutral Discussion Bacterial bron chial infection is the most endopeptidase enhances the effect of FMLP on common precip: itant of acute respiratory failure cholinergic neurotransmission in rabbit airway in subjects with chronic airflow limitation'2 and in vitro25 though we found a pronounced a major cause oif morbidity and mortality. We reduction in the bronchoconstrictor response found that sub Ijects with chronic bronchitis to FMLP inhalation after inhibition of neutral bronchoconstri(cted to lower doses of inhaled endopeptidase invivo.26Neutral endopeptidase FMLP and re covery from FMLP induced activity does not decline with age and, if bronchoconstril ction was delayed in the older anything, serum activity is increased modestly subjects irrespe ctive of the presence of chronic in subjects with chronic airflow limitation.27 The prolonged response to FMLP in the older bronchitis. There was n lo difference in PD20 FMLP, subjects cannot be explained by changes in recovery from bronchoconstriction or in the neutral endopeptidase. The neutrophil dependent oedema seen in haematological effect of FMLP inhalation between the exc-smokers and those who had rabbit skin after FMLP injection is markedly never smoked iin the control group, so we feel accentuated by vasodilator pretreatment and that they repres gent a valid control group. The has a halflife of 40 minutes.28 Persistence of differences in IFMLP responses between the airway oedema could explain the prolonged bronchitic sut bjects and the ex-smoking effect of FMLP in older subjects but whether controls occurre .d despite a close match for total clearance of oedema slows with age or smoking historry. This implies that excess bronchoconstriction is related more closely to sputum producition predicts greater sensitivity airway oedema in this group is unknown. to FMLP inhal; ation. Leucocyte depletion'5 and anticholinergic Small airwalys of smokers with airflow agents23 inhibit FMLP induced bronchoconobstruction hav7e thickened walls and smaller striction but there is no evidence of differences luminal diame ters than those of control in the cholinergic or leucocyte response to subjects.'7 As a result, the reduction in airflow FMLP in this age group to account for the would be expectted to be greater in patients with slower recovery. FMLP inhalation did not produce changes chronic bronchiitis for an equivalent degree of smooth muscle contraction. If this was the sole in neutrophil or monocyte counts in venous White cells
12
Neutrophils
Downloaded from http://thorax.bmj.com/ on March 24, 2015 - Published by group.bmj.com
Effects of inhalation of N-formyl-methionyl-leucyl-phenylalanine
blood in either group of the magnitude that we had seen in normal subjects.29 There was also a total absence of facial flushing in both older groups after FMLP inhalation whereas flushing is seen in over 75% of young subjects,""293' always occurring after the first FMLP dose and always associated with marked neutropenia.29 Transient neutropenia after FMLP inhalation probably reflects synchronous activation of circulating neutrophils and temporary sequestration in the microcirculation. We have usually given a single inhalation of 0-4 jumol FMLP to young subjects after finding variability in the neutrophil responses to incremental doses of FMLP (0 1, 0-5, 1 Mmol).3` Nevertheless, all young normal subjects inhaling the incremental doses of FMLP had either a transient neutropenia or neutrophilia or both; in this study even the five older control subjects who had a dose of FMLP equivalent to that given to young normal subjects had a minimal neutrophil leucocytosis following FMLP. The leucocyte response to FMLP may require access of FMLP to the circulation. Neutrophils from well older subjects have higher resting intracellular calcium concentrations and the response to a variety of stimuli including FMLP is slightly reduced.32 33 A final possibility is that the neutrophils of older subjects, behave in a similar way to those of younger subjects but less in synchrony so that flushing and profound neutropenia which may require synchronous neutrophil activation adherence are not seen. Although the neutropenic response was absent in the older subjects, bronchoconstriction was similar in the older control subjects to that in the younger normal subjects and was increased in subjects with chronic bronchitis. The role that leucocytes play in FMLP induced bronchoconstriction is clearly not linked directly to the development of neutropenia in either age group. In this study, subjects with chronic bronchitis showed greater sensitivity to the effects of inhaled FMLP and older subjects showed delayed recovery from FMLP induced bronchoconstriction in comparison with young subjects. The combination of disease related increased sensitivity and age related slowing of recovery would place an older patient with chronic bronchitis at special risk ofthe effects of FMLP and related formyl peptides produced by bacteria during bacterial bronchial infection. We thank Boehringer Ingelheim and the Asthma Foundation of New South Wales for their support. 1 Burk RH, George RB. Acute respiratory failure in chronic obstructive pulmonary disease. Arch Intern Med 1973; 132:865-8. 2 Dardes N, Campo S, Chiappini MG, Re MA, Cicciarell P, Vulterini S. Prognosis of COPD patients after an episode of acute respiratory failure. Eur J Respir Dis 1986;69 (Suppl 146).377-81. 3 Martin RR, White A. The in vitro release of leukocyte histamine by staphylococcal antigens. J Immunol 1969; 102:437-41. 4 Van der Zwan JC, Orie NGM, Kauffman HF, Wiers PWJ, De Vries K. Bronchial obstructive reactions after inhalation with endotoxin and precipitogens of Haemophilus influenzae in patients with chronic non-specific lung disease. Clin Allergy 1982;12:547-9. 5 Simonsson BG, Svedmyr N, Skoogh BE, Andersson R, Bergh NP. In vivo and in vitro studies on alpha-receptors in human airways; potentiation with bacterial endotoxin. Scand J Respir Dis 1972;53:227-36.
283 6 Adams JM, Capecchi MR. N-formylmethionyl-sRNA as the initiator of protein synthesis. Proc Natl Acad Sci USA 1966;55: 147-54. 7 Capecchi MR. Initiation of E. coli proteins. Proc Natl Acad Sci USA 1966;55:1517-24. 8 Becker EL. The formylpeptide receptor of the neutrophil. Am JPathol 1987;129:16-24. 9 Carp H. Mitochondrial N-formylmethionyl proteins as chemoattractants for neutrophils. J Exp Med 1982;155: 264-75. 10 Armour CL, Black JL, Johnson PRA, Vincenc KS, Berend N. Formylpeptide induced contraction of human airways in vitro. J Appl Physiol 1986;60:141-6. 11 Berend N, Peters MJ, Armour CL, Black JL, Ward HE. Effect of formyl-methionyl-leucyl-phenylalanine on airway function. Thorax 1988;42:36-40. 12 Peters MJ, Panaretto K, Breslin ABX, Berend N. Effects of prolonged inhalation of N-formyl-methionyl-leucylphenylalanine on airway function in rabbits. J Appl Physiol 1991;70:2448-54. 13 Mullen JBM, Wright JL, Wiggs BR, Pare PD, Hogg JC. Reassessment of inflammation in airways in chronic bronchitis. BMJ 1985;291:1235-40. 14 Jubiz W, Radmark C, Malmsten C, et al. A novel leukotriene produced by stimulation of leukocytes with formylmethionylleucylphenylalanine. J Biol Chem 1982;257: 6106-10. 15 Berend N, Armour CL, Black JL. Formyl-methionylleucyl-phenylalanine causes bronchocontriction in rabbits. Agents Actions 1986;17:466-71. 16 Yan K, Salome C, Woolcock AJ. Rapid method for measurement of histamine responsiveness. Thorax 1983; 38:760-5. 17 Bosken C, Wiggs B, Pare P, Hogg J. Small airways dimensions in smokers with obstruction to airflow. Am Rev Respir Dis 1990;142:563-70. 18 Zimmerli W, Seligmann BE, Gallin JI. Neutrophils are hyperpolarised after exudation and show an increased response to formyl-peptide but not to phorbol myristyl acetate. Eur J Clin Invest 1987;17:435-41. 19 Zimmerli W, Seligmann BE, Gallin JI. Exudation primes human and guinea-pig neutrophils for subsequent responsiveness to the chemotactic peptide fMet-Leu-Phe and increases C3bi receptor expression. J Clin Invest 1986;77:925-33. 20 Williams JD, Robin J-L, Lewis RA, Lee TH, Austen KF. Generation of leukotrienes by human monocytes pretreated with cytochalasin B and stimulated with formylmethionyl-leucyl-phenyalalanine. J Immunol 1986;136: 642-8. 21 Boukili MA, Bureau M-F, Lellouch-Tubiana A, Lefort J, Simon M-T, Varhaftig BB. Alveolar macrophages and eicosanoids but not neutrophils mediate bronchoconstriction induced by FMLP in the guinea-pig. Br J Pharmacol 1989;98:61-70. 22 Hobson CH, Butt TJ, Ferry DM, Hunter J, Chadwick VS, Broom MF. Enterohepatic recirculation of bacterial chemotactic peptide in rats with experimental colitis. Gastroenterology 1988;94:1006-13. 23 Llorens C, Schwartz J-C. Enkephalinase activity in rat peripheral organs. Eur J Pharmacol 1981;69:113-16. 24 Connelly JC, Skidgel RA, Schulz WW, Johnson AR, Erdos EG. Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide. Proc Natl Acad Sci USA 1985;82:8737-41. 25 Kanemura T, Tamaoki J, Sakai N, Kobayashi K, Takizawa T. The effect ofN-formyl-methionyl-leucylphenylalanine on cholinergic neurotransmission and its modulation by enkephalinase in rabbit airway smooth muscle. Reg Pept 1989;26: 107-16. 26 Peters MJ, Panaretto K, Breslin ABX, Berend N. Effect of neutral endopeptidase inhibition on f-Met-Leu-Phe induced bronchoconstriction in the rabbit. J Appl Physiol 1991;70:877-81. 27 Johnson AR, Coalson JL, Ashton J, Larumbride M, Erdos EG. Neutral endopeptidase in serum samples from patients with adult respiratory distress syndrome. Comparison with angiotensin converting enzyme. Am Rev Respir Dis 1985;132:1262-7. 28 Hellewell PG, Yarwood H, Williams TJ. Characteristics of oedema formation induced by N-formyl-methionylleucyl-phenylalanine in rabbit skin. Br J Pharmacol 1989; 97:181-9. 29 Peters MJ, Breslin ABX, Kemp AS, Chu J, Berend N. The hematologicaleffectsofinhalationofN-formyl-methionylleucyl-phenylalanine in man. Thorax 1992;47:284-7. 30 Peters MJ, Breslin ABX, Berend N. The effect of anticholinergic and beta-agonist pretreatment on bronchoconstriction induced by N-formyl-methionyl-leucylphenylalanine. Fur Respir I 1989,2.464-9. 31 Peters MJ, Karet B, Breslin ABX, Berend N. Histamine reactivity and hematologic changes following N-formylmethionyl-leucyl-phenylalanine (FMLP) inhalation in man [abstract]. Aust NZ J Med 1987;17(Suppl):520. 32 Varga Z, Kovacs EM, Paragh G, Jacob M-P, Robert L, Fulop T. Effects of elastin peptides and N-formylmethionyl-leucyl-phenyalanine on cytosolic free calcium in polymorphonuclear leukocytes in healthy middle-aged and elderly subjects. Clin Biochem 1988;21:127-30. 33 Lipschitz DA, Udupa KB, Boxer LA. The role ofcalcium in the age related decline of neutrophil function. Blood 1988;71:659-65.
Downloaded from http://thorax.bmj.com/ on March 24, 2015 - Published by group.bmj.com
Effects of inhalation of N-formyl-methionyl-leucyl-phenylalanine in the well elderly and in patients with chronic bronchitis. M J Peters, A B Breslin and N Berend Thorax 1992 47: 279-283
doi: 10.1136/thx.47.4.279 Updated information and services can be found at: http://thorax.bmj.com/content/47/4/279
These include:
Email alerting service
Receive free email alerts when new articles cite this article. Sign up in the box at the top right corner of the online article.
Notes
To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions To order reprints go to: http://journals.bmj.com/cgi/reprintform To subscribe to BMJ go to: http://group.bmj.com/subscribe/
